Ecological Questions 28 (2017) 4: 41–46 http://dx.doi.org/10.12775/EQ.2017.037

Adhesive and hydrophobic properties of and Pseudomonas cedrina associated with cosmetics

-XOLD=DELHOVND*$OLQD.XQLFND6W\F]\ĔVND$QQD2WOHZVND

,QVWLWXWHRI)HUPHQWDWLRQ7HFKQRORJ\DQG0LFURELRORJ\)DFXOW\RI%LRWHFKQRORJ\DQG)RRG6FLHQFHV Lodz University of Technology, Wolczanska 171/173 St, 90-924 Lodz, Poland, *e-mail: [email protected] Received: 08 October 2017 /Accepted: 24 November 2017

$EEUHYLDWLRQV TSB, Trypticase Soya Broth; PBS, phosphate buffered saline; TSA, Trypticase Soya Agar; PUM, phosphate urea magnesium sulfate buffer; ATTC, American Type Culture Collection; DSM, Deutsche Sammlung von Mikroorganismen; ISO, Inter- national Organization for Standardization; RAPEX, Rapid Alert System.

$EVWUDFW The presence of in the cosmetic production environment is often connected with non-sterile raw materials, inap- propriate production lines disinfection or cross contamination. Among bacteria isolated from the environment, opportunistic can be also found, posing a risk to patients with lowered immunity. Moreover, their susceptibility to antibiotics and disinfectants is frequently decreased as they develop more complex forms - biofilms. As hydrophobicity and adhesive properties play a vital role in the colonization process the aim of this research was to determine hydrophobic, aggregative and adhesive properties of bacteria isolated from the cosmetics. Bacteria used in the research were isolated from the body balm and the cosmetic preservative (three strains of Pseudomonas aeru- ginosa and four strains of Pseudomonas cedrina DQGLGHQWLILHGXVLQJ6U51$JHQHVHTXHQFLQJ)RUWKRVHVWUDLQVDQGDOVRWZR reference strains (P. aeruginosa ATCC15442 and P. cedrina DSM17516) an aggregation test, hydrophobicity by two different methods (SAT and MATH) and adhesion to polystyrene by crystal violet binding assay were performed. According to the SAT method more than half of the tested strains were strongly hydrophobic. Using MATH test, it was proved that four strains (P. cedrina DSM17516 and three isolates of P. aeruginosa) were strong hydrophobes, however, the rest of the strains expressed moderate hydrophobicity. Moreover, self-aggregation was also observed and for P. aeruginosa&),,ZDVPRUHWKDQ All of the strains were able to adhere to polystyrene after 30 minutes contact, almost all of them (excluding P. cedrina DSM17516) indicated a moderate adhesion already after four hours of incubation. These results indicate that environmental Pseudomonas strains possess strong hydrophobic and adhesive properties, that may results in a colonization of abiotic surfaces.

.H\ZRUGVPseudomonas, cosmetic contamination, hydrophobicity, adhesion, biofilm.

,QWURGXFWLRQ also from the production line due to itsimproper disinfec- tion and cross-contaminations. Those irregularities may Cosmetics may be the source of microorganisms, both lead to biodeterioration of products, revealing a change pathogenic and non-pathogenic. Apart from the secondary of pH, color, smell, consistency, viscosity, phase separation contamination in the course of usage, microbial contamina- DQGJDVLILFDWLRQ *DUEROLĔVND  tion may take also place during the production processes. Pseudomonas sp. is a group of Gram-negative bacte- Microorganisms may be introduced withraw materials and ria, frequently causing nosocomial infections of patients 42 Julia Zabielska, Alina Kunicka-Styczyńska, Anna Otlewska

with reduced immunity. P. aeruginosa belonging to this GATCCTGGCTCAG-3’) and 1492r (5’-GGTTACCTT- group is one of the common species associated with infec- GTTACGACTT-3’). PCR reactions were performed using tions and are usually very tough to eradicate. Among many 1.5 U RedTaqReadyMix DNA polymerase (Sigma-Al- virulence factors of P. aeruginosa the ones connected with GULFK86$ ȝ/RIHDFKSULPHUVROXWLRQ ȝ0 DQG the process of adhesion and biofilm formation (capabil- ȝ/RIWHPSODWH'1$ QJȝ/ LQWKH0-0LQL*UD- ity of aggregation, cell hydrophobicity, rhamnolipid and dient Thermal Cycler (Bio-Rad, USA). The amplification EPS production, motility etc.) can be (Briendstien et al., SDUDPHWHUVZHUHXVHGDVSUHYLRXVO\GHVFULEHG.UĊJLHOHWDO 2011). The ability of colonization of abiotic surfaces by P. (2014). The PCR fragments were purified using Clean Up aeruginosa SRO\PHUPDWHULDOVZDVUHSRUWHGE\=DELHOVND 0LQL.LW $ $%LRWHFKQRORJ\3RODQG 7KH6U51$ et al. (2015) and the biofilm formed reached more than 6 sequences were obtained using the BigDye Terminator ORJ&)8FP2 after 24 hours of incubation. Once the biofilm 5HDG\ 5HDFWLRQ &\FOH 6HTXHQFLQJ .LW $SSOLHG %LRV\V- is developed, its removing is much more complicated due tems, USA) and analyzed with the Applied Biosystems to numerous mechanisms such as the presence of matrix model 3730 Genetic Analyzer (Genomed S.A., Poland). or “super resistant” cells – metabolically inactive, grown The nucleotide sequences of 16S rRNA were proofread, in the nutrition and oxygen deficiency (Matusiak, 2014). assembled and compared with sequences available in The The purpose of this study was to identify microorgan- National Center for Biotechnology Information (NCBI) us- isms isolated from a cosmetic product and a cationic sur- LQJEDVLFORFDODOLJQPHQWVHDUFKWRRO%/$67 =KDQJ factant used as a cosmetic ingredient and determine their et al., 2010). The nucleotide sequences of 16S rRNA gene hydrophobic, aggregative and adhesive properties, crucial were deposited in GenBank of the NCBI under the fol- for the biofilm development. ORZLQJDFFHVVLRQQXPEHUV.<.< P. ce- drina DQG.<.< P. aeruginosa).

0DWHULDODQGPHWKRGV 3K\ORJHQHWLFDQDO\VLV 0LFURRUJDQLVPV The phylogenetic tree was constructed by neighbor-joining method using the MEGA7 software (Saitou & Nei, 1987; A total of seven strains were isolated: three P. aeruginosa .XPDUHWDO 7KHHYROXWLRQDU\GLVWDQFHVZHUHFRP- &),,&),9D&)9 IURPDERG\EDOPDQGIRXUEHORQJLQJ puted using the Maximum Composite Likelihood method to P. cedrina (DH1, DH2, DN3, DH4) from cetyltrimethyl and are in the units of the number of base substitutions ammonium chloride, a commercial cationic surfactant used per site (Tamura et al., 2004). The analysis involved 13 as a cosmetic ingredient. One ml of appropriate product nucleotide sequences of 16S rRNA genes from the NCBI was diluted in 10 ml of saline solution with Tween 80 to database. All positions containing gaps and missing data neutralize the sample. One ml of each sample was spread were eliminated. There was a total of 1384 positions in on cetrimide agar (BTL, Poland) to allow Pseudomonas the final dataset. sp. growth. Strains were initially identified by biochemi- FDOWHVWV$3,( ELR0pULHX[)UDQFH $GGLWLRQDOO\WZR $JJUHJDWLRQWHVW reference strains were used – P. aeruginosa ATCC15442 (American Type Culture Collection) and P. cedrina Aggregation of strains was evaluated by the method pro- DSM17516 (Deutsche Sammlung von Mikroorganismen). posed by Del Re et al. (2000). Bacteria were grown in TSB Bacteria were activated in TSB (Merck, Germany) for 24 (Merck, Germany) for 24 hours at 30ºC. Two ml of the KRXUV DW ž& )RU SK\ORJHQHWLF DQDO\VLV WKH VHTXHQFHV culture was centrifuged for 10 minutes (5000 rpm). The of the following reference strains were used: P. cedrina precipitate was suspended in two ml of 10 μM PBS buffer C37, G25, CI-10 originated from milk and clausen (CI-10); and the absorbance at the wavelength 600 nm was meas- P. aeruginosa=0.8-0)4,,,RULJLQDWHGUHVSHFWLYH- ured (A). Subsequently, the suspension was incubated for O\IURPWH[WLOHZDVWHZDWHU.DO\DQLVHZDJHWUHDWPHQWSODQW two hours at 30ºC and the absorbance of the upper layer and sputum. was measured (A0). Aggregation was calculated according to the formula: ,GHQWLILFDWLRQRIEDFWHULDOVWUDLQV Bacterial strains were identified based on 16S rRNA gene sequencing. Briefly, genomic DNA was extracted employ-   LQJWKH*HQRPLF0LQL.LW $ $%LRWHFKQRORJ\3RODQG  according to manufacturer’s protocol. 16S rRNA gene was amplified with universal primers 27f (5’-AGAGTTT- Adhesive and hydrophobic properties of Pseudomonas aeruginosa 43

+\GURSKRELFLW\±0$7+DQG6$7DVVD\ the plates were again rinsed with water. After drying, each well was decolorized with 96% ethanol and the absorb- Hydrophobic properties of the cell surface were deter- DQFH ZDV PHDVXUHG Ȝ  QP 7KH EODQN VDPSOH ZDV mined by two different methods SAT (Salt Aggregation the growth medium. Test) and MATH (Microbial Adhesion to Hydrocarbon). In the SAT method, activated bacteria were inoculat- 6WDWLVWLFDODQDO\VLV ed on TSA (Merck, Germany) and incubated for 24 hours at 30ºC. After that, bacteria inoculum in 2 μM phosphate The tests were conducted in triplicate and SD (standard buffer saline (PBS, Merck, Germany) was prepared (ap- deviation) was calculated using ORIGIN LAB 6.1 version SUR[žLQ0F)DUODQGVFDOH DQGPL[HGZLWKDPPRQLXP (Northampton, USA). The correlation between buffers sulfate of different molarity (from 0.1 to 4) in a ratio 1:1. used in MATH method was evaluated by Pearson’s coef- The control included bacteria inoculum in PBS buffer. Af- ficient analysis. ter 15–20 minutes, the aggregates formation was evalu- ated by macroscopic observations and classified according to the scale proposed by Nwanyanwu and Abu (2013), in 5HVXOWVDQGGLVFXVVLRQ which strains were divided into three categories: of high hydrophobicity (<1.0 M), of moderate hydrophobicity Seven strains belonging to Pseudomonas species were iso- (1.0–2.0 M) and of low hydrophobicity (>2.0 M). lated from the body balm and the cosmetic preservative. In the MATH assay with some modifications (Rosen- Genetic identification based on 16S rRNA gene sequenc- berg, 1984), bacteria were activated on TSA slants (in- LQJVKRZHGWKDWWKUHHVWUDLQV &),,&),9DDQG&)9 DUH cubation 24 hours, 30ºC). Then, inoculum (A1) was pre- classified as P. aeruginosa and four (DH1, DH2, DH3 and pared either in 10 μM phosphate buffer saline (PBS), or in DH4) as P. cedrina. Phylogenetic analysis, presented in phosphate urea magnesium sulfate buffer (PUM, Merck, WKH)LJXUHVKRZHGDVLPLODULW\WRWKHW\SHVWUDLQV Germany). Two and half ml of inoculum was mixed with The presence of unfavorable microflorawas described half ml of p-xylene and incubated for 10 minutes at 37ºC. also by Campana et al. (2006). They investigated 91 com- Thereafter, samples were homogenized and incubated once mercial cosmetics and opportunistic strains such as Pseu- again for 45 minutes. The samples were allowed to be sep- domonas putida and Staphylococcus aureus were found in arated into two phases (aqueous and hydrocarbon). The some samples. According to the different source - RAPEX aqueous phase (A2) was measured spectrophotometrically data for non-food products in the EU, only in the last dec- at the wavelength 520 nm. The hydrophobicity index (per- ade over 100 cases of microbiological contaminations were centage of cell adhesion to hydrocarbon) was calculated noted, including the presence of opportunistic pathogens upon the following formula: such as P. aeruginosa, Burkholderia cepacia and S. aureus. Also Birketsoz et al. (2013) indicated the contamination of P. aeruginosa, P. putida and Pseudomonas fluorescens in commercial, unopened cosmetic products.  In the course of the study aggregative, adhesive and hydrophobic properties of the identified Pseudomonas sp. 7KHUHVXOWVZHUHHYDOXDWHGDFFRUGLQJWRWKHVFDOH .DG- strains were assessed. Aggregation and hydrophobic abili- am et al., 2009): strongly hydrophobic (>50%), moderate ties expressed by Pseudomonas strains were presented in hydrophobic (20–50%) and low hydrophobic (<20%). 7DEOHDQG)LJXUH7KHKLJKHVWYDOXHVRIDJJUHJDWLRQ was presented by environmental P. aeruginosa VWUDLQ&),, $GKHVLRQWRSRO\VW\UHQH and it reached 20.74%. The aggregation of the majority of strains fluctuated from 9 to 13%. The least aggregative Adhesion to polystyrene was carried out by the method strain was the reference strain P. aeruginosa ATCC15442 proposed by Stepanovic et al. (2000) with some modifica- (4.82%). Aggregation of different pathogens such as Sal- tions. Bacteria were cultivated in TSB (Merck, Germany) monella typhimurium, Listeria monocytogenes, Staphylo- for 24 hours at 30ºC. 20 μl of bacteria was carried into coccus aureus and was described by Xu 230 μl of TSB on 96-well microtitration plate. Plates were et al. (2010). The values obtained by Xu et al. (l.c.) were incubated in different periods of time – for 0.5, 4 and 24 similar and oscillated from 5 to 23% for bacteria in PBS hours, then the content was poured out and wells were buffer, however after addition of NaCl in different con- rinsed with sterile water. The plates were dried and adhered centration aggregation were much higher. The aggrega- bacteria cells were fixed with 96% ethanol for 20–30 min- tion is connected with cell-to-cell binding and as it was utes. Afterwards, ethanol was removed and bacteria were showed, environmental conditions have a great influence stained with 0.5% crystal violet. The dye was removed and on it. 44 Julia Zabielska, Alina Kunicka-Styczyńska, Anna Otlewska

)LJXUH3K\ORJHQHWLFWUHHRIPseudomonas sp. cosmetic contaminant: P. aeruginosa &),,&),9D&)9LVRODWHGIURPDERG\EDOP P. cedrina DH1, DH2, DN3, DH4 isolated from cetyltrimethyl ammonium chloride; P. cedrina C37, G25, CI-10 origi- nated from milk and clausen (CI-10); P. aeruginosa=0.8-0)4,,,RULJLQDWHGUHVSHFWLYHO\IURPWH[WLOHZDVWHZDWHU .DO\DQLVHZDJHWUHDWPHQWSODQWDQGVSXWXP

Hydrophobicity of isolated bacteria was determined Table 1. Pseudomonas sp. strains aggregation and degree of cell by two methods. The first one – Salt Aggregation Test surface hydrophobicity assessed by *SAT method – indicated that all of the strains were high or moderate hydrophobes. In comparison to environmental isolates, $JJUHJDWLRQ +\GURSKR Microorganism reference strains were less hydrophobic (Table 1). The (%) bicity* same method was used by Wolska et al. (2002) for clini- cal strains of P. aeruginosa. More than half of the tested DSM17516 11.57±3.64 moderate bacteria expressed strong hydrophobic properties. Result of Pseudomonas sp. hydrophobicity using MATH method DH1 11.80±3.43 high ZHUHSUHVHQWHGLQ)LJXUHP. cedrina was characterized by Pseudomonas cedrina DH2 10.50±4.11 moderate the highest hydrophobicity index, which reached respec- tively 88.74% in PBS buffer and 89.62% in PUM buffer, DH3 9.43±0.86 high and was classified as strongly hydrophobic. P. aeruginosa DH4 10.58±1.47 high LVRODWHV &) H[SUHVVHGDOVRDVWURQJK\GURSKRELFLW\ZLWK index values oscillated between 60–80%. The lowest index ATCC15442 4.82±0.11 moderate values were obtained for P. cedrina strains (DH 1–4) and &),, 20.74±1.66 high for the reference strain P. aeruginosa ATCC15442, what Pseudomonas classified them as moderate hydrophobes. aeruginosa &),9D 12.94±0.47 high Moreover, the degree of hydrophobicity evaluated by &)9 11.00±1.87 moderate Pearson’s coefficient analysis showed strong correlation Adhesive and hydrophobic properties of Pseudomonas aeruginosa 45

between both buffers tested. Norouzi et al. (2010) also pre- The crystal violet binding method was used to assess sented data regarding P. aeruginosa strains hydrophobicity Pseudomonas sp. strains capability of adhering to polysty- assessed by the MATH method and the majority of strains rene and creating biofilm, thus to evaluate their adhesive demonstrated moderate hydrophobic properties. Research SURSHUWLHV )LJ $OORIWKHVWUDLQVZHUHDEOHWRDGKHUHWR FRQGXFWHGE\.DGDPHWDO  RQHQYLURQPHQWDOVWUDLQV the polystyrene surface after 0.5 hour contact and the high- P. aeruginosa and P. fluorescens indicated that both strains est absorbance values were reached for P. cedrina isolates are moderate hydrophobic in PBS buffer (28.8% and 36.4% (DH1–4). After 4 hours of contact for almost all strains respectively) and low hydrophobic in PUM buffer (18% and absorbance ranged from 0.12 to 0.22 (excluding P. cedrina 15.2% respectively). Tyfa et al. (2015) tested Gram-positive DSM17516), which indicated on their moderate adherence. Alicyclobacillus sp. using the same method and almost all 24-hour incubation showed that strains P. cedrina: DH3, of them were strong hydrophobes in PUM buffer and ma- DH1 and P. aeruginosa ATCC15442 expressed a great ad- jority of strains were moderate hydrophobes in PBS buffer. KHVLRQ)RUP. cedrina DSM17516 reference strain the ab-

)LJXUHPseudomonas sp. cosmetic contaminants hydrophobicity index estimated by the MATH test in PBS and PUMbuffers, clas- sified as: S – strongly hydrophobic, M – moderate hydrophobic and L – low hydrophobic

)LJXUHPseudomonas sp. cosmetic contaminants adhesion to polystyrene within 0.5–24 hours of incubation 46 Julia Zabielska, Alina Kunicka-Styczyńska, Anna Otlewska

sorbance reached 0.097 which indicated on weak adhesion. NUXVWDFMLFHZQLNDXURORJLF]QHJR>8ULQDU\WUDFWLQIHF- The rest of strains expressed moderate adhesion. Similar re- tions caused by – role of the biofilm sults to P. cedrina '06ZDVUHSRUWHGE\6HGOiþNRYi DQGWKHHQFUXVWDWLRQRIWKHXURORJLFDOFDWKHWHU@3RVWĊS\ et al. (2011) for Pseudomonas fluorescens reaching after Mikrobiologii 53(2): 173–181. 0.5 hour incubation the absorbance equal 0.087. Nwanyanwu C. & Abu G., 2013, Influence of growth All in all, cosmetics can be the source of opportunistic media on hydrophobicity of phenol-utilizing bacteria pathogens which pose a health threat to humans with im- found in petroleum refinery effluent. International Re- paired immune system. In pursuance of the ISO standard search Journal of Biological Sciences 2: 6–10. (PN-EN ISO 17516: 2014-11), the presence of microbes 1RURX]L)0DQVRXUL60RUDGL0 5D]DYL0 such as P. aeruginosa, S. aureus and Candida albicans is Comparison of cell surface hydrophobicity and bio- not allowed in one gram of the cosmetic product, however film formation among ESBL- and non-ESBL-produc- these microorganisms are often found in cosmetics or their ing Pseudomonas aeruginosa clinical isolates. African ingredients, which was proved in this report. What is more, Journal of Microbiology Research 4(11): 1143–1147. such bacteria often present a great hydrophobic and adhe- PN-EN ISO 17516: 2014-11 Cosmetics-Microbiology-Mi- sive properties which can lead to colonization of abiotic crobiological limits. surfaces and biofilms creation. Rosenberg M., 1984, Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydropho- ELFLW\)(060LFURELRORJ\/HWWHUV± 5HIHUHQFHV Saitou N. & Nei M., 1987, The neighbor-joining method: A new method for reconstructing phylogenetic trees. Birteksoz A.R., Tuysuz M. & Otuk G., 2013, Investiga- Molecular Biology and Evolution 4: 406–425. tion of preservative efficacy and microbiological con- 6HGOiþNRYi3ýHĜRYVNê0+RUViNRYi, 9ROGĜLFK0 tent of some cosmetics found on the market. Pakistan 2011, Cell surface characteristic of bogorensis – Journal of Pharmaceutical Sciences 26: 153–157. spoilage microorganism of bottled water. Czech Journal %ULHGHQVWHLQ(%0'HOD)XHQWH1XQH]& +DQFRFN RI)RRG6FLHQFHV  ± R.E.W., 2011, Pseudomonas aeruginosa: all roads lead 6WHSDQRYLF69XNRYLF''DNLF,6DYLF% 6YDELF to resistance. Trends in Microbiology 19: 419–426. 9ODKRYLF 0  $ PRGLILHG PLFURWLWHUSODWH WHVW &DPSDQD56FHVD&3DWURQH99LWWRULD( %DIIRQH for quantification of staphylococcal biofilm formation. W., 2006, Microbiological study of cosmetics product Journal of Microbiological Methods 40(2): 175–179. during their use by consumers: health risk and efficacy 7DPXUD.1HL0 .XPDU63URVSHFWVIRULQ- of preservative systems. Letters in Applied Microbiol- ferring very large phylogenies by using the neighbor- ogy 43: 301–306. joining method. Proceedings of the National Academy Del Re B., Sgorbati B., Miglioli M. & Palenzona D., 2000, of Sciences (USA) 101:11030–11035. Adhesion, autoaggregation and hydrophobicity of 13 7\ID $ .XQLFND6W\F]\ĔVND $  =DELHOVND -  strains of Bifidobacterium longum. Letters in Applied Evaluation of hydrophobicity of quantitative analysis Microbiology 31: 438–442. of biofilm formation by Alicyclobacillus sp. Acta Bio- *DUEROLĔVND00LFURELRORJLFDOFRQWUROLQWKHFRV- chimica Polonica 62(4): 785–790. PHWLF LQGXVWU\ ĝZLDW 3U]HP\VáX .RVPHW\F]QHJR  ;X+=RX</HH+< $KQ-(IIHFWRI1D&O 26–29. on the biofilm formation by foodborne pathogens. Jour- .DGDP7$5XSD/%DOKDO'.7RWHZDG1' *\D- QDORI)RRG6FLHQFH  ± nanath G., 2009, Determination of the degree of hydro- :ROVND.3RJRU]HOVND6)LMRá(-DNXEF]DN$ %X- phobicity – A technique to assess bacterial colonization NRZVNL .  ,QIOXHQFH RQ JURZWK FRQGLWLRQV RQ on leaf surface and root region of lotus plant. Asian cell surface hydrophobicity of Pseudomonas aerugi- Journal of Experimental Sciences 23(1): 135–139. nosa 0HG\F\QD 'RĞZLDGF]DOQD L 0LNURELRORJLD  .UĊJLHO'2WOHZVND$ $QWRODN+$WWDFKPHQW 61–66. of Asaia bogorensis originating in fruit-flavored water =DELHOVND - .XQLFND6W\F]\ĔVND $ 5DMNRZVND . to packaging materials. BioMed Research Internation- & Tyfa A., 2015, Opportunistic Gram-negative rods’ al. (http://dx.doi.org/10.1155/2014/514190). capability of creating biofilm structures on polivynyl .XPDU66WHFKHU* 7DPXUD.0(*$0R- chloride and styrene-acronitrile copolymer surfaces. lecular Evolutionary Genetics Analysis version 7.0 for Acta Biochimica Polonica 62(4): 733–737. bigger datasets. Molecular Biology and Evolution 33: =KDQJ = 6FKZDUW] 6 :DJQHU /  0LOOHU :  1870–1874. A greedy algorithm for aligning DNA sequences. Jour- 0DWXVLDN '0  =DNDĪHQLD XNáDGX PRF]RZHJR nal of Computational Biology 7: 203–214. ] XG]LDáHP Proteus mirabilis – rola biofilmu i in-